Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several AIDS-related cancers including Kaposi's sarcoma and primary effusion lymphoma. Despite intensive studies, the mechanisms underlying KSHV oncogenesis and persistent infection remain unclear. KSHV encodes more than two dozens microRNAs (miRs) derived from 12 precursor miRs. Others and we have shown that KSHV miRs regulate cell growth and survival, enhance cell invasion and angiogenesis, evade host immune responses, and promote viral latency. However, most of these studies have been carried out by overexpressing miRs without taking into consideration of KSHV infection. The objective of Project 3 is to identify the specific miRs and mechanisms that mediate KSHV oncogenesis and persistent infection in the context of viral infection. Our preliminary results have shown that a cluster of 10 KSHV pre-miRs is required for KSHV cellular transformation of primary mesenchymal stem cells (MSCs). Furthermore, we have shown that KSHV miR-K1 activates the NF- KB pathway and inhibits lytic replication by targeting IKBO while miR-KIO variants inhibit TGF-P pathway to block apoptosis by targeting TGF-(5 type II receptor. Therefore, our working hypothesis is that specific KSHV mlRs manipulate essential cellular pathways and key viral genes, contributing critically to KSHV oncogenesis and persistent infection. We have developed several novel systems that can address these challenges including model of KSHV cell growth transformation and tumorigenesis, model of KSHV infection in NOD/SCID lL2Ry-/- (NSG) humanized mice, KSHV reverse genetics system, and transcription activator-like effector nucleases (TALEN)-mediated genome editing technology. We will carry out the following three integrated and synergistic Specific Aims: 1. To identify KSHV essential miRs for cell growth transformation and tumorigenesis; 2. To delineate the mechanisms by which KSHV miRs regulate oncogenesis; and 3. To identify KSHV essential miRs for persistent infection in NSG humanized mice. The proposed works are highly significant because they will, for the first time, define the functions and mechanisms of action of KSHV miRs in oncogenesis and persistent infection using innovative approaches and newly developed model systems. The study will establish a novel paradigm of oncogenesis mediated by viral subversion ofthe mlR pathway, thus providing insights into developing innovative therapeutic methods for KSHV-induced cancers and understanding the oncogenesis of other cancers.